The present invention relates to bioanalytical methods and compositions; and more particularly it relates to improved fluorometric assay methods and fluorochrome compositions for selectively evaluating DNA content of cells or for screening for compounds that interact with crude, partially purified and purified DNA.
Various pathological conditions and chemotherapeutic regimens suppress lymphocyte activation. For example, patients with cancer, immunodeficiency diseases, organ grafts, autoimmune disease, and acute drug toxicity may exhibit an inhibition of lymphocyte activation. As a consequence of suppressed lymphocyte activation, these patients also incur increased susceptibility to serious infection, even susceptibility to ordinarily innocuous fungi, bacteria and viruses. The chronic effects of suppressed lymphocyte activation are correlated with a higher incidence of developing various cancers, particularly lymphomas and sarcomas. Furthermore, in patients with existing specific types of cancer, autoimmune disorders and infections, the intercurrent impairments of lymphocyte responsiveness correlate respectively with enhanced rate of tumor growth, exacerbation of autoimmune pathology and accelerated infection. In some cases, these invader-host imbalances are produced by soluble mediators synthesized and released into patient sera by the tumors, autoimmune tissues and infectious agents themselves.
One reliable, in vitro, measure of lymphocyte activation is the degree of DNA synthesis that occurs within the cell population following stimulation with an appropriately presented antigen, mitogen (lectin), host mediator, pharmaceutical or environmental agent. Deoxyribonucleic acid (DNA) is a complex and intricate nucleoprotein which through messenger RNA, provides the genetic coding for enzymes, antibodies and other cell proteins. Suppressed lymphocytes, both T and B cells, almost always exhibit a corresponding suppression of spontaneous and/or stimulated DNA synthesis.
At present, the principal in vitro test for assessment of lymphocyte activation involves a microtiter culture procedure using tritiated thymidine, a radiolabelled precurser of DNA or other cellular components, which become incorporated into lymphocyte DNA and other cellular metabolites, affording an approximate measure of the rate of DNA synthesis. Several problems, however exist with implementation of this technique as a diagnostic bioanalytical assay. A primary drawback of the conventional .sup.3 H-thymidine assay is the susceptibility of cellular uptake, metabolism, and eventual incorporation of the .sup.3 H-thymidine into DNA to interference by multiple normal and abnormal serum components, test substances and byproducts which have either no effects or no directly proportional effects on DNA synthesis. Often false results are obtained because soluble test substances released by necrotic tumor cells from cancer patients, macrophages from autoimmune patients and experimental animals, abnormal lymphoid tissues from immunodeficient patients, and mass production commercial systems for natural products such as hybridoma cultures and bacterial fermentation vats include interfering substances such as "cold nucleosides" which compete for the .sup.3 H-thymidine uptake into cellular components. Hence, the nonspecificity of .sup.3 H-thymidine as an indicator of DNA synthetic rates results in false, high or low, indications of lymphocyte activation.
Furthermore, the .sup.3 H-thymidine assay test suffers from the disadvantages inherently connected with radioisotopic materials and techniques, such as their incumbent expense, health hazards, waste disposal problems and laboratory licensing requirements.
Accordingly, there remains a need for a more specific assay for lymphocyte activation, and modulators thereof, than the .sup.3 H-thymidine assay. It would be advantageous to provide such methods that eliminate both artifactual suppression and enhancement of lymphocyte activation which are commonly produced by thymidine, adenosine, deoxyadenosine and other cold nucleosides, ribavirin, vincristine, vinblastine, colchicine and other natural and synthetic products.
In addition to overcoming or circumventing the many drawbacks associated with prior art lymphocyte activation assays, the present invention also offers a selective screening system for substances that may interact with DNA.
Many compounds which directly interact with, bind to or alter the conformation of DNA also exhibit mutagenic, carcinogenic and/or immunotoxicant activities. Furthermore, many antineoplastic agents modulate cell replication by binding to DNA thereby altering its primary structure, destabilizing the double helix and/or depolymerizing the polynucleotide sequence.
Recently, fluorescence polarization techniques and instruments have been developed to compare the DNA binding characteristics of chemotherapeutic agents (such as actinomycin) and carcinogenic agents by competitive binding against the fluorescent, intercalating probe, acridine orange. While fluorescence polarization represents an elegant approach to the screening of DNA-interactive compounds that constitute potentional carcinogens and antitumor agents, instrument expense and difficulties achieving the modes of automation required to process large sample numbers have precluded its widespread industrial, commercial, and clinical application. Moreover, many of the fluorochrome systems employed are not specific for DNA, but rather exhibit binding activity with other related nucleotides including synthetic monostranded nucleoprotein, noneucaryotic nucleotides and RNA.
Accordingly, it would be advantageous to provide a rapid, automated inexpensive procedure that is based on fluorescence enhancement rather than polarization, and that can utilize standard fluorometers as well as polarization type fluorometers for the detection and quantitative determination of compounds which bind selectively to DNA. Further, it would be advantageous that such procedures utilize composition and mixtures having long shelf lives which concommitantly afford the sensitivity required for routine lymphocyte activation assays employed by clinical, commercial and basic research laboratories.